Project Summary: Persistent pain is a long-term condition that adversely affects psychological, physical and social aspects of patients. Current treatment methods rely heavily on opioids and anti-inflammatory drugs which can lose efficacy over time or produce deleterious side effects. Persistent pain patients often experience static or dynamic mechanical allodynia which is a painful sensation caused by innocuous stimuli that arises from punctate indentation of the skin or light brushing across the skin surface. Mechanisms that convert light touch into pain in the setting of injury occur within the spinal dorsal horn. Identifying the neural circuitry involved and developing ways to target it therapeutically are of intense interest. Our laboratory recently identified a subset of cholecystokinin (CCK) expressing excitatory interneurons that are important for conveying both static and dynamic mechanical allodynia. These neurons reside in the low threshold mechanosensory zone (lamina IIi-IV) of the dorsal horn. Their role in both forms of mechanical allodynia induced by inflammatory and neuropathic injuries was demonstrated by acute silencing of these neurons using a virally targeted Designer Receptor Exclusively Activated by Designer Drugs (DREADD). Heat hypersensitivity in the inflammatory model was also reversed. Interestingly, neurons that transiently express the vesicular glutamate transporter 3 (VGLUT3) during the first two weeks of postnatal development are a subset of the lamina III-IV CCK population and are important only for conveying dynamic allodynia. To identify more precisely which CCK subset is responsible for the different forms of persistent pain, we are utilizing a single-cell transcriptome study of mouse dorsal horn, which reported that the CCK population is composed of three molecularly distinct subsets marked by Maf, Cpne4 and Trh. In preliminary experiments, I have identified the CCK neurons that we have targeted with the DREADD as expressing either Trh or Cpne4. Since all of the Trh+ neurons were included in the targeted population, in Aim 1a, I will determine the role of Trh+ neurons in persistent pain. Since the transient VGLUT3 neurons are a subpopulation of the CCK neurons and are required only for dynamic allodynia, in Aim 1b, I will similarly determine the identity of these cells. To better understand clinically relevant persistent pain circuitry, the laboratory would like to apply the same tools used in mouse to the macaque and eventually to human. Thus, In Aim 2, I will determine in the macaque dorsal horn, the molecular identity and laminar organization of the CCK subsets as a percentage of excitatory neurons within each lamina. Conservation of molecular profiles and anatomic distribution of cell-types between the mouse and non-human primate would be suggestive of a conservation of their functional roles. The work will provide important information for the design of novel viral strategies to target cell-types in the primate dorsal horn to study pain circuits as well as develop novel, non- opioid based therapeutics for mechanical allodynia.